Aerodynamically light particles for pulmonary drug delivery

ABSTRACT

Improved aerodynamically light particles for drug delivery to the pulmonary system, and methods for their synthesis and administration are provided. In a preferred embodiment, the aerodynamically light particles are made of a biodegradable material and have a tap density less than 0.4 g/cm 3  and a mass mean diameter between 5 μm and 30 μm. The particles may be formed of biodegradable materials such as biodegradable polymers. For example, the particles may be formed of a functionalized polyester graft copolymer consisting of a linear α-hydroxy-acid polyester backbone having at least one amino acid group incorporated therein and at least one poly(amino acid) side chain extending from an amino acid group in the polyester backbone. In one embodiment, aerodynamically light particles having a large mean diameter, for example greater than 5 μm, can be used for enhanced delivery of a therapeutic agent to the alveolar region of the lung. The aerodynamically light particles incorporating a therapeutic agent may be effectively aerosolized for administration to the respiratory tract to permit systemic or local delivery of wide variety of therapeutic agents.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Divisional application of U.S. application Ser.No. 08/739,308, filed on Oct. 29, 1996, now U.S. Pat. No. 5,874,064,which is a Continuation-in-Part of U.S. application Ser. No. 08/655,570,filed on May 24, 1996, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates generally to biodegradable particles oflow density and large size for drug delivery to the pulmonary system.

Biodegradable particles have been developed for the controlled-releaseand delivery of protein and peptide drugs. Langer, R., Science, 249:1527-1533 (1990). Examples include the use of biodegradable particlesfor gene therapy (Mulligan, R. C. Science, 260: 926-932 (1993)) and for`single-shot` immunization by vaccine delivery (Eldridge et al., Mol.Immunol., 28: 287-294 (1991)).

Aerosols for the delivery of therapeutic agents to the respiratory tracthave been developed. Adjei, A. and Garren, J. Pharm. Res. 7, 565-569(1990); and Zanen, P. and Lamm, J.-W. J. Int. J. Pharm. 114, 111-115(1995). The respiratory tract encompasses the upper airways, includingthe oropharynx and larynx, followed by the lower airways, which includethe trachea followed by bifurcations into the bronchi and bronchioli.The upper and lower airways are called the conducting airways. Theterminal bronchioli then divide into respiratory bronchioli which thenlead to the ultimate respiratory zone, the alveoli, or deep lung. Gonda,I. "Aerosols for delivery of therapeutic and diagnostic agents to therespiratory tract," in Critical Reviews in Therapeutic Drug CarrierSystems 6:273-313, 1990. The deep lung, or alveoli, are the primarytarget of inhaled therapeutic aerosols for systemic drug delivery.

Inhaled aerosols have been used for the treatment of local lungdisorders including asthma and cystic fibrosis (Anderson et al., Am.Rev. Respir. Dis., 140: 1317-1324 (1989)) and have potential for thesystemic delivery of peptides and proteins as well (Patton and Platz,Advanced Drug Delivery Reviews, 8:179-196 (1992)). However, pulmonarydrug delivery strategies present many difficulties for the delivery ofmacromolecules; these include protein denaturation duringaerosolization, excessive loss of inhaled drug in the oropharyngealcavity (often exceeding 80%), poor control over the site of deposition,irreproducibility of therapeutic results owing to variations inbreathing patterns, the often too-rapid absorption of drug potentiallyresulting in local toxic effects, and phagocytosis by lung macrophages.

Considerable attention has been devoted to the design of therapeuticaerosol inhalers to improve the efficiency of inhalation therapies.Timsina et. al., Int. J. Pharm. 101, 1-13 (1995); and Tansey, I. P.,Spray Technol. Market 4, 26-29 (1994). Attention has also been given tothe design of dry powder aerosol surface texture, regarding particularlythe need to avoid particle aggregation, a phenomenon which considerablydiminishes the efficiency of inhalation therapies owing to particleaggregation. French, D. L., Edwards, D. A. and Niven, R. W., J. AerosolSci. 27, 769-783 (1996). Attention has not been given to the possibilityof using large particle size (>5 μm) as a means to improveaerosolization efficiency, despite the fact that intraparticle adhesiondiminishes with increasing particle size. French, D. L., Edwards, D. A.and Niven, R. W. J. Aerosol Sci. 27, 769-783 (1996). This is becauseparticles of standard mass density (mass density near 1 g/cm³) and meandiameters >5 μm are known to deposit excessively in the upper airways orthe inhaler device. Heyder, J. et al., J. Aerosol Sci., 17: 811-825(1986). For this reason, dry powder aerosols for inhalation therapy aregenerally produced with mean diameters primarily in the range of <5 μm.Ganderton, D., J. Biopharmaceutical Sciences 3:101-105 (1992); andGonda, I. "Physico-Chemical Principles in Aerosol Delivery," in Topicsin Pharmaceutical Sciences 1991, Crommelin, D. J. and K. K. Midha, Eds.,Medpharm Scientific Publishers, Stuttgart, pp. 95-115, 1992. Large"carrier" particles (containing no drug) have been co-delivered withtherapeutic aerosols to aid in achieving efficient aerosolization amongother possible benefits. French, D. L., Edwards, D. A. and Niven, R. W.J. Aerosol Sci. 27, 769-783 (1996).

Local and systemic inhalation therapies can often benefit from arelatively slow controlled release of the therapeutic agent. Gonda, I.,"Physico-chemical principles in aerosol delivery," in: Topics inPharmaceutical Sciences 1991, D. J. A. Crommelin and K. K. Midha, Eds.,Stuttgart: Medpharm Scientific Publishers, pp. 95-117, (1992). Slowrelease from a therapeutic aerosol can prolong the residence of anadministered drug in the airways or acini, and diminish the rate of drugappearance in the bloodstream. Also, patient compliance is increased byreducing the frequency of dosing. Langer, R., Science, 249:1527-1533(1990); and Gonda, I. "Aerosols for delivery of therapeutic anddiagnostic agents to the respiratory tract," in Critical Reviews inTherapeutic Drug Carrier Systems 6:273-313, (1990).

The human lungs can remove or rapidly degrade hydrolytically cleavabledeposited aerosols over periods ranging from minutes to hours. In theupper airways, ciliated epithelia contribute to the "mucociliaryescalator" by which particles are swept from the airways toward themouth. Pavia, D. "Lung Mucociliary Clearance," in Aerosols and the Lung:Clinical and Experimental Aspects, Clarke, S. W. and Pavia, D., Eds.,Butterworths, London, 1984. Anderson et al., Am. Rev. Respir. Dis., 140:1317-1324 (1989). In the deep lungs, alveolar macrophages are capable ofphagocytosing particles soon after their deposition. Warheit, M. B. andHartsky, M. A., Microscopy Res. Tech. 26: 412-422 (1993); Brain, J. D.,"Physiology and Pathophysiology of Pulmonary Macrophages," in TheReticuloendothelial System, S. M. Reichard and J. Filkins, Eds., Plenum,New York, pp. 315-327, 1985; Dorries, A. M. and Valberg, P. A., Am. Rev.Resp. Disease 146, 831-837 (1991); and Gehr, P. et al. Microscopy Res.and Tech., 26, 423-436 (1993). As the diameter of particles exceeds 3μm, there is increasingly less phagocytosis by macrophages. Kawaguchi,H. et al., Biomaterials 7: 61-66 (1986); Krenis, L. J. and Strauss, B.,Proc. Soc. Exp. Med., 107:748-750 (1961); and Rudt, S. and Muller, R.H., J. Contr. Rel., 22: 263-272 (1992). However, increasing the particlesize also minimizes the probability of particles (possessing standardmass density) entering the airways and acini due to excessive depositionin the oropharyngeal or nasal regions. Heyder, J. et al., J. AerosolSci., 17: 811-825 (1986). An effective dry-powder inhalation therapy forboth short and long term release of therapeutics, either for local orsystemic delivery, requires a powder that displays minimum aggregation,as well as a means of avoiding or suspending the lung's naturalclearance mechanisms until drugs have been effectively delivered.

There is a need for improved inhaled aerosols for pulmonary delivery oftherapeutic agents. There is a need for the development of drug carrierswhich are capable of delivering the drug in an effective amount into theairways or the alveolar zone of the lung. There further is a need forthe development of drug carriers for use as inhaled aerosols which arebiodegradable and are capable of controlled release of drug within theairways or in the alveolar zone of the lung.

It is therefore an object of the present invention to provide improvedcarriers for the pulmonary delivery of therapeutic agents. It is afurther object of the invention to provide inhaled aerosols which areeffective carriers for delivery of therapeutic agents to the deep lung.It is another object of the invention to provide carriers for pulmonarydelivery which avoid phagocytosis in the deep lung. It is a furtherobject of the invention to provide carriers for pulmonary drug deliverywhich are capable of biodegrading and releasing the drug at a controlledrate.

SUMMARY OF THE INVENTION

Improved aerodynamically light particles for drug delivery to thepulmonary system, and methods for their synthesis and administration areprovided. In a preferred embodiment, the particles are made of abiodegradable material, have a tap density less than 0.4 g/cm³ and amean diameter between 5 μm and 30 μm. In one embodiment, for example, atleast 90% of the particles have a mean diameter between 5 μm and 30 μm.The particles may be formed of biodegradable materials such asbiodegradable polymers, proteins, or other water-soluble materials. Forexample, the particles may be formed of a functionalized polyester graftcopolymer consisting of a linear α-hydroxy-acid polyester backbonehaving at least one amino acid residue incorporated per molecule thereinand at least one poly(amino acid) side chain extending from an aminoacid group in the polyester backbone. Other examples include particlesformed of water-soluble excipients, such as trehalose or lactose, orproteins, such as lysozyme or insulin. The aerodynamically lightparticles can be used for enhanced delivery of a therapeutic agent tothe airways or the alveolar region of the lung. The particlesincorporating a therapeutic agent may be effectively aerosolized foradministration to the respiratory tract to permit systemic or localdelivery of a wide variety of therapeutic agents. They optionally may beco-delivered with larger carrier particles, not carrying a therapeuticagent, which have for example a mean diameter ranging between about 50μm and 100 μm.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph comparing total particle mass of aerodynamically lightand non-light, control particles deposited on the nonrespirable andrespirable stages of a cascade impactor following aerosolization.

FIG. 2 is a graph comparing total particle mass deposited in the tracheaand after the carina (lungs) in rat lungs and upper airways followingintratracheal aerosolization during forced ventilation ofaerodynamically light PLAL-Lys particles and control, non-light PLAparticles.

FIG. 3 is a graph comparing total particle recovery of aerodynamicallylight PLAL-Lys particles and control PLA particles in rat lungs andfollowing bronchoalveolar lavage.

DETAILED DESCRIPTION OF THE INVENTION

Aerodynamically light, biodegradable particles for improved delivery oftherapeutic agents to the respiratory tract are provided. The particlescan be used in one embodiment for controlled systemic or local drugdelivery to the respiratory tract via aerosolization. In a preferredembodiment, the particles have a tap density less than about 0.4 g/cm³.Features of the particle which can contribute to low tap density includeirregular surface texture and porous structure. Administration of thelow density particles to the lung by aerosolization permits deep lungdelivery of relatively large diameter therapeutic aerosols, for example,greater than 5 μm in mean diameter. A rough surface texture also canreduce particle agglomeration and provide a highly flowable powder,which is ideal for aerosolization via dry powder inhaler devices,leading to lower deposition in the mouth, throat and inhaler device.

Density and Size of Aerodynamically Light Particles

Particle Size

The mass mean diameter of the particles can be measured using a CoulterCounter. The aerodynamically light particles are preferably at leastabout 5 microns in diameter. The diameter of particles in a sample willrange depending upon depending on factors such as particle compositionand methods of synthesis. The distribution of size of particles in asample can be selected to permit optimal deposition within targetedsites within the respiratory tract.

The aerodynamically light particles may be fabricated or separated, forexample by filtration, to provide a particle sample with a preselectedsize distribution. For example, greater than 30%, 50%, 70%, or 80% ofthe particles in a sample can have a diameter within a selected range ofat least 5 μm. The selected range within which a certain percentage ofthe particles must fall may be for example, between about 5 and 30 μm,or optionally between 5 and 15 μm. In one preferred embodiment, at leasta portion of the particles have a diameter between about 9 and 11 μm.Optionally, the particle sample also can be fabricated wherein at least90%, or optionally 95% or 99%, have a diameter within the selectedrange. The presence of the higher proportion of the aerodynamicallylight, larger diameter (at least about 5 μm) particles in the particlesample enhances the delivery of therapeutic or diagnostic agentsincorporated therein to the deep lung.

In one embodiment, in the particle sample, the interquartile range maybe 2 μm, with a mean diameter for example of 7.5, 8.0, 8.5, 9.0, 9.5,10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0 or 13.5 μm. Thus, for example,at least 30%, 40%, 50% or 60% of the particles may have diameters withinthe selected range 5.5-7.5 μm, 6.0-8.0 μm, 6.5-8.5 μm, 7.0-9.0 μm,7.5-9.5 μm, 8.0-10.0 μm, 8.5-10.5 μm, 9.0-11.0 μm, 9.5-11.5 μm,10.0-12.0 μm, 10.5-12.5 μm, 11.0-13.0 μm, 11.5-13.5 μm, 12.0-14.0 μm,12.5-14.5 μm or 13.0-15.0 μm. Preferably the said percentages ofparticles have diameters within a 1 μm range, for example, 6.0-7.0 μm,10.0-11.0 μm or 13.0-14.0 μm.

The aerodynamically light particles incorporating a therapeutic drug,and having a tap density less than about 0.4 g/cm³, with mean diametersof at least about 5 μm, are more capable of escaping inertial andgravitational deposition in the oropharyngeal region, and are targetedto the airways or the deep lung. The use of larger particles (meandiameter at least about 5 μm) is advantageous since they are able toaerosolize more efficiently than smaller, non-light aerosol particlessuch as those currently used for inhalation therapies.

In comparison to smaller non-light particles, the larger (at least about5 μm) aerodynamically light particles also can potentially moresuccessfully avoid phagocytic engulfment by alveolar macrophages andclearance from the lungs, due to size exclusion of the particles fromthe phagocytes' cytosolic space. Phagocytosis of particles by alveolarmacrophages diminishes precipitously as particle diameter increasesbeyond 3 μm. Kawaguchi, H. et al., Biomaterials 7: 61-66 (1986); Krenis,L. J. and Strauss, B., Proc. Soc. Exp. Med., 107:748-750 (1961); andRudt, S. and Muller, R. H., J. Contr. Rel., 22: 263-272 (1992). Forparticles of statistically isotropic shape (on average, particles of thepowder possess no distinguishable orientation), such as spheres withrough surfaces, the particle envelope volume is approximately equivalentto the volume of cytosolic space required within a macrophage forcomplete particle phagocytosis.

Aerodynamically light particles thus are capable of a longer termrelease of a therapeutic agent. Following inhalation, aerodynamicallylight biodegradable particles can deposit in the lungs (due to theirrelatively low tap density), and subsequently undergo slow degradationand drug release, without the majority of the particles beingphagocytosed by alveolar macrophages. The drug can be deliveredrelatively slowly into the alveolar fluid, and at a controlled rate intothe blood stream, minimizing possible toxic responses of exposed cellsto an excessively high concentration of the drug. The aerodynamicallylight particles thus are highly suitable for inhalation therapies,particularly in controlled release applications. The preferred meandiameter for aerodynamically light particles for inhalation therapy isat least about 5 μm, for example between about 5 and 30 μm.

The particles may be fabricated with the appropriate material, surfaceroughness, diameter and tap density for localized delivery to selectedregions of the respiratory tract such as the deep lung or upper airways.For example, higher density or larger particles may be used for upperairway delivery, or a mixture of different sized particles in a sample,provided with the same or different therapeutic agent may beadministered to target different regions of the lung in oneadministration.

Particle Density and Deposition

The particles having a diameter of at least about 5 μm and incorporatinga therapeutic or diagnostic agent preferably are aerodynamically light.As used herein, the phrase "aerodynamically light particles" refers toparticles having a tap density less than about 0.4 g/cm³. The tapdensity of particles of a dry powder may be obtained using a GeoPyc™(Micrometrics Instrument Corp., Norcross, Ga. 30093). Tap density is astandard measure of the envelope mass density. The envelope mass densityof an isotropic particle is defined as the mass of the particle dividedby the minimum sphere envelope volume within which it can be enclosed.

Inertial impaction and gravitational settling of aerosols arepredominant deposition mechanisms in the airways and acini of the lungsduring normal breathing conditions. Edwards, D. A., J. Aerosol Sci.26:293-317 (1995). The importance of both deposition mechanismsincreases in proportion to the mass of aerosols and not to particle (orenvelope) volume. Since the site of aerosol deposition in the lungs isdetermined by the mass of the aerosol (at least for particles of meanaerodynamic diameter greater than approximately 1 μm), diminishing thetap density by increasing particle surface irregularities and particleporosity permits the delivery of larger particle envelope volumes intothe lungs, all other physical parameters being equal.

The low tap density particles have a small aerodynamic diameter incomparison to the actual envelope sphere diameter. The aerodynamicdiameter, d_(aer), is related to the envelope sphere diameter, d (Gonda,I., "Physico-chemical principles in aerosol delivery," in Topics inPharmaceutical Sciences 1991 (eds. D. J. A. Crommelin and K. K. Midha),pp. 95-117, Stuttgart: Medpharm Scientific Publishers, 1992) by theformula:

    d.sub.aer =d√ρ

where the envelope mass ρ is in units of g/cm³. Maximal deposition ofmonodisperse aerosol particles in the alveolar region of the human lung(˜60%) occurs for an aerodynamic diameter of approximately d_(aer) =3μm. Heyder, J. et al., J. Aerosol Sci., 17: 811-825 (1986). Due to theirsmall envelope mass density, the actual diameter d of aerodynamicallylight particles comprising a monodisperse inhaled powder that willexhibit maximum deep-lung deposition is:

    d=3/√ρ μm (where ρ<1 g/cm.sup.3);

where d is always greater than 3 μm. For example, aerodynamically lightparticles that display an envelope mass density, ρ=0.1 g/cm³, willexhibit a maximum deposition for particles having envelope diameters aslarge as 9.5 μm. The increased particle size diminishes interparticleadhesion forces. Visser, J., Powder Technology, 58:1-10. Thus, largeparticle size increases efficiency of aerosolization to the deep lungfor particles of low envelope mass density, in addition to contributingto lower phagocytic losses.

Particle Materials

In order to serve as efficient and safe drug carriers in drug deliverysystems, the aerodynamically light particles preferably arebiodegradable and biocompatible, and optionally are capable ofbiodegrading at a controlled rate for delivery of a drug. The particlescan be made of any material which is capable of forming a particlehaving a tap density less than about 0.4 g/cm³. Both inorganic andorganic materials can be used. For example, ceramics may be used. Othernon-polymeric materials (e.g. fatty acids) may be used which are capableof forming aerodynamically light particles as defined herein. Differentproperties of the particle can contribute to the aerodynamic lightnessincluding the composition forming the particle, and the presence ofirregular surface structure or pores or cavities within the particle.

Polymeric Particles

The particles may be formed from any biocompatible, and preferablybiodegradable polymer, copolymer, or blend, which is capable of formingparticles having a tap density less than about 0.4 g/cm³.

Surface eroding polymers such as polyanhydrides may be used to form theaerodynamically light particles. For example, polyanhydrides such aspoly[(p-carboxyphenoxy)-hexane anhydride] (PCPH) may be used.Biodegradable polyanhydrides are described, for example, in U.S. Pat.No. 4,857,311, the disclosure of which is incorporated herein byreference.

In another embodiment, bulk eroding polymers such as those based onpolyesters including poly(hydroxy acids) can be used. For example,polyglycolic acid (PGA) or polylactic acid (PLA) or copolymers thereofmay be used to form the aerodynamically light particles, wherein thepolyester has incorporated therein a charged or functionalizable groupsuch as an amino acid as described below.

Other polymers include polyamides, polycarbonates, polyalkylenes such aspolyethylene, polypropylene, poly(ethylene glycol), poly(ethyleneoxide), poly(ethylene terephthalate), poly vinyl compounds such aspolyvinyl alcohols, polyvinyl ethers, and polyvinyl esters, polymers ofacrylic and methacrylic acids, celluloses and other polysaccharides, andpeptides or proteins, or copolymers or blends thereof which are capableof forming aerodynamically light particles with a tap density less thanabout 0.4 g/cm³. Polymers may be selected with or modified to have theappropriate stability and degradation rates in vivo for differentcontrolled drug delivery applications.

Polyester Graft Copolymers

In one preferred embodiment, the aerodynamically light particles areformed from functionalized polyester graft copolymers, as described inHrkach et al., Macromolecules, 28:4736-4739 (1995); and Hrkach et al.,"Poly(L-Lactic acid-co-amino acid) Graft Copolymers: A Class ofFunctional Biodegradable Biomaterials" in Hydrogels and BiodegradablePolymers for Bioapplications, ACS Symposium Series No. 627, Raphael M.Ottenbrite et al., Eds., American Chemical Society, Chapter 8, pp.93-101, 1996, the disclosures of which are incorporated herein byreference. The functionalized graft copolymers are copolymers ofpolyesters, such as poly(glycolic acid) or poly(lactic acid), andanother polymer including functionalizable or ionizable groups, such asa poly(amino acid). In a preferred embodiment, comb-like graftcopolymers are used which include a linear polyester backbone havingamino acids incorporated therein, and poly(amino acid) side chains whichextend from the amino acid residues in the polyester backbone. Thepolyesters may be polymers of α-hydroxy acids such as lactic acid,glycolic acid, hydroxybutyric acid and hydroxy valeric acid, orderivatives or combinations thereof. The inclusion of ionizable sidechains, such as polylysine, in the polymer has been found to enable theformation of more aerodynamically light particles, using techniques formaking microparticles known in the art, such as solvent evaporation.Other ionizable groups, such as amino or carboxyl groups, may beincorporated, covalently or noncovalently, into the polymer to enhancesurface roughness and porosity. For example, polyalanine could beincorporated into the polymer.

An exemplary polyester graft copolymer, which may be used to formaerodynamically light polymeric particles is the graft copolymer,poly(lactic acid-co-lysine-graft-lysine) (PLAL-Lys), which has apolyester backbone consisting of poly(L-lactic acid-co-L-lysine) (PLAL),and grafted poly-lysine chains. PLAL-Lys is a comb-like graft copolymerhaving a backbone composition, for example, of 98 mol % lactic acid and2 mol % lysine and poly(lysine) side chains extending from the lysinesites of the backbone.

PLAL-Lys may be synthesized as follows. First, the PLAL copolymerconsisting of L-lactic acid units and approximately 1-2% N εcarbobenzoxy-L-lysine (Z-L-lysine) units is synthesized as described inBarrera et al., J. Am. Chem. Soc., 115:11010 (1993). Removal of the Zprotecting groups of the randomly incorporated lysine groups in thepolymer chain of PLAL yields the free ε-amine which can undergo furtherchemical modification. The use of the poly(lactic acid) copolymer isadvantageous since it biodegrades into lactic acid and lysine, which canbe processed by the body. The existing backbone lysine groups are usedas initiating sites for the growth of poly(amino acid) side chains.

The lysine ε-amino groups of linear poly(L-lactic acid-co-L-lysine)copolymers initiate the ring opening polymerization of an amino acid N-εcarboxyanhydride (NCA) to produce poly(L-lactic acid-co-amino acid)comb-like graft copolymers. In a preferred embodiment, NCAs aresynthesized by reacting the appropriate amino acid with triphosgene.Daly et al., Tetrahedron Lett., 29:5859 (1988). The advantage of usingtriphosgene over phosgene gas is that it is a solid material, andtherefore, safer and easier to handle. It also is soluble in THF andhexane so any excess is efficiently separated from the NCAs.

The ring opening polymerization of amino acid N-carboxyanhydrides (NCAs)is initiated by nucleophilic initiators such as amines, alcohols, andwater. The primary amine initiated ring opening polymerization of NCAsallows good control over the degree of polymerization when the monomerto initiator ratio (M/I) is less than 150. Kricheldorf, H. R. in Modelsof Biopolymers by Ring-Opening Polymerization, Penczek, S., Ed., CRCPress, Boca Raton, 1990, Chapter 1; Kricheldorf, H. R.α-Aminoacid-N-Carboxy-Anhydrides and Related Heterocycles,Springer-Verlag, Berlin, 1987; and Imanishi, Y. in Ring-OpeningPolymerization, Ivin, K. J. and Saegusa, T., Eds., Elsevier, London,1984, Volume 2, Chapter 8. Methods for using lysine ε-amino groups aspolymeric initiators for NCA polymerizations are described in the art.Sela, M. et al., J. Am. Chem. Soc., 78: 746 (1956).

In the reaction of an amino acid NCA with PLAL, the nucleophilic primaryε-amino of the lysine side chain attacks C-5 of the NCA leading to ringopening and formation of the amino acid amide, along with the evolutionof CO₂. Propagation takes place via further attack of the amino group ofthe amino acid amides on subsequent NCA molecules. The degree ofpolymerization of the poly(amino acid) side chains, the correspondingamino acid content in the graft copolymers and their resulting physicaland chemical characteristics can be controlled by changing the M/I ratiofor the NCA polymerization--that is, changing the ratio of NCA to lysineε-amino groups of pLAL. Thus, in the synthesis, the length of thepoly(amino acid), such as poly(lysine), side chains and the total aminoacid content in the polymer may be designed and synthesized for aparticular application.

The poly(amino acid) side chains grafted onto or incorporated into thepolyester backbone can include any amino acid, such as aspartic acid,alanine or lysine, or mixtures thereof. The functional groups present inthe amino acid side chains, which can be chemically modified, includeamino, carboxylic acid, thiol, guanido, imidazole and hydroxyl groups.As used herein, the term "amino acid" includes natural and syntheticamino acids and derivatives thereof. The polymers can be prepared with arange of amino acid side chain lengths, for example, about 10-100 ormore amino acids, and with an overall amino acid content of, forexample, 7-72% or more depending on the reaction conditions. Thegrafting of poly(amino acids) from the pLAL backbone may be conducted ina solvent such as dioxane, DMF, or CH₂ Cl₂, or mixtures thereof. In apreferred embodiment, the reaction is conducted at room temperature forabout 2-4 days in dioxane.

Alternatively, the aerodynamically light particles for pulmonary drugdelivery may be formed from polymers or blends of polymers withdifferent polyester/amino acid backbones and grafted amino acid sidechains. For example, poly(lactic acid-co-lysine-graft-alanine-lysine)(PLAL-Ala-Lys), or a blend of PLAL-Lys with poly(lactic acid-co-glycolicacid-block-ethylene oxide) (PLGA-PEG) (PLAL-Lys-PLGA-PEG) may be used.

In the synthesis, the graft copolymers may be tailored to optimizedifferent characteristics of the aerodynamically light particleincluding: i) interactions between the agent to be delivered and thecopolymer to provide stabilization of the agent and retention ofactivity upon delivery; ii) rate of polymer degradation and, thereby,rate of drug release profiles; iii) surface characteristics andtargeting capabilities via chemical modification; and iv) particleporosity.

Formation of Aerodynamically Light Polymeric Particles

Aerodynamically light polymeric particles may be prepared using singleand double emulsion solvent evaporation, spray drying, solventextraction and other methods well known to those of ordinary skill inthe art. The aerodynamically light particles may be made, for exampleusing methods for making microspheres or microcapsules known in the art.

Methods developed for making microspheres for drug delivery aredescribed in the literature, for example, as described by Mathiowitz andLanger, J. Controlled Release 5,13-22 (1987); Mathiowitz, et al.,Reactive Polymers 6, 275-283 (1987); and Mathiowitz, et al., J. Appl.Polymer Sci. 35, 755-774 (1988), the teachings of which are incorporatedherein. The selection of the method depends on the polymer selection,the size, external morphology, and crystallinity that is desired, asdescribed, for example, by Mathiowitz, et al., Scanning Microscopy4,329-340 (1990); Mathiowitz, et al., J. Appl. Polymer Sci. 45, 125-134(1992); and Benita, et al., J. Pharm. Sci. 73, 1721-1724 (1984), theteachings of which are incorporated herein.

In solvent evaporation, described for example, in Mathiowitz, et al.,(1990), Benita, and U.S. Pat. No. 4,272,398 to Jaffe, the polymer isdissolved in a volatile organic solvent, such as methylene chloride.Several different polymer concentrations can be used, for example,between 0.05 and 0.20 g/ml. The drug, either in soluble form ordispersed as fine particles, is added to the polymer solution, and themixture is suspended in an aqueous phase that contains a surface activeagent such as poly(vinyl alcohol). The aqueous phase may be, forexample, a concentration of 1% poly(vinyl alcohol) w/v in distilledwater. The resulting emulsion is stirred until most of the organicsolvent evaporates, leaving solid microspheres, which may be washed withwater and dried overnight in a lyophilizer.

Microspheres with different sizes (1-1000 microns) and morphologies canbe obtained by this method which is useful for relatively stablepolymers such as polyesters and polystyrene. However, labile polymerssuch as polyanhydrides may degrade due to exposure to water. For thesepolymers, solvent removal may be preferred.

Solvent removal was primarily designed for use with polyanhydrides. Inthis method, the drug is dispersed or dissolved in a solution of aselected polymer in a volatile organic solvent like methylene chloride.The mixture is then suspended in oil, such as silicon oil, by stirring,to form an emulsion. Within 24 hours, the solvent diffuses into the oilphase and the emulsion droplets harden into solid polymer microspheres.Unlike solvent evaporation, this method can be used to make microspheresfrom polymers with high melting points and a wide range of molecularweights. Microspheres having a diameter for example between one and 300microns can be obtained with this procedure.

Targeting of Particles

Targeting molecules can be attached to the aerodynamically lightparticles via reactive functional groups on the particles. For example,targeting molecules can be attached to the amino acid groups offunctionalized polyester graft copolymer particles, such as PLAL-Lysparticles. Targeting molecules permit binding interaction of theparticle with specific receptor sites, such as those within the lungs.The particles can be targeted by attachment of ligands whichspecifically or non-specifically bind to particular targets. Exemplarytargeting molecules include antibodies and fragments thereof includingthe variable regions, lectins, and hormones or other organic moleculescapable of specific binding for example to receptors on the surfaces ofthe target cells.

Therapeutic Agents

Any of a variety of therapeutic, prophylactic or diagnostic agents canbe incorporated within the aerodynamically light particles. Theaerodynamically light particles can be used to locally or systemicallydeliver a variety of therapeutic agents to an animal. Examples includesynthetic inorganic and organic compounds, proteins and peptides,polysaccharides and other sugars, lipids, and nucleic acid sequenceshaving therapeutic, prophylactic or diagnostic activities. Nucleic acidsequences include genes, antisense molecules which bind to complementaryDNA to inhibit transcription, and ribozymes. The agents to beincorporated can have a variety of biological activities, such asvasoactive agents, neuroactive agents, hormones, anticoagulants,immunomodulating agents, cytotoxic agents, prophylactic agents,antibiotics, antivirals, antisense, antigens, and antibodies. In someinstances, the proteins may be antibodies or antigens which otherwisewould have to be administered by injection to elicit an appropriateresponse. Compounds with a wide range of molecular weight can beencapsulated, for example, between 100 and 500,000 grams per mole.

Proteins are defined as consisting of 100 amino acid residues or more;peptides are less than 100 amino acid residues. Unless otherwise stated,the term protein refers to both proteins and peptides. Examples includeinsulin and other hormones. Polysaccharides, such as heparin, can alsobe administered.

The aerodynamically light polymeric aerosols are useful as carriers fora variety of inhalation therapies. They can be used to encapsulate smalland large drugs, release encapsulated drugs over time periods rangingfrom hours to months, and withstand extreme conditions duringaerosolization or following deposition in the lungs that might otherwiseharm the encapsulated therapeutic.

The aerodynamically light particles may include a therapeutic agent forlocal delivery within the lung, such as agents for the treatment ofasthma, emphysema, or cystic fibrosis, or for systemic treatment. Forexample, genes for the treatment of diseases such as cystic fibrosis canbe administered, as can beta agonists for asthma. Other specifictherapeutic agents include, but are not limited to, insulin, calcitonin,leuprolide (or LHRH), G-CSF, parathyroid hormone-related peptide,somatostatin, testosterone, progesterone, estradiol, nicotine, fentanyl,norethisterone, clonidine, scopolomine, salicylate, cromolyn sodium,salmeterol, formeterol, albeterol, and vallium.

Administration

The particles including a therapeutic agent may be administered alone orin any appropriate pharmaceutical carrier, such as a liquid, for examplesaline, or a powder, for administration to the respiratory system. Theycan be co-delivered with larger carrier particles, not including atherapeutic agent, the latter possessing mass mean diameters for examplein the range 50 μm-100 μm.

Aerosol dosage, formulations and delivery systems may be selected for aparticular therapeutic application, as described, for example, in Gonda,I. "Aerosols for delivery of therapeutic and diagnostic agents to therespiratory tract," in Critical Reviews in Therapeutic Drug CarrierSystems, 6:273-313, 1990; and in Moren, "Aerosol dosage forms andformulations," in: Aerosols in Medicine. Principles, Diagnosis andTherapy, Moren, et al., Eds, Esevier, Amsterdam, 1985, the disclosuresof which are incorporated herein by reference.

The greater efficiency of aerosolization by aerodynamically lightparticles of relatively large size permits more drug to be deliveredthan is possible with the same mass of non-light aerosols. Therelatively large size of aerodynamically light aerosols depositing inthe deep lungs also minimizes potential drug losses caused by particlephagocytosis. The use of aerodynamically light polymeric aerosols astherapeutic carriers provides the benefits of biodegradable polymers forcontrolled release in the lungs and long-time local action or systemicbioavailability. Denaturation of macromolecular drugs can be minimizedduring aerosolization since macromolecules are contained and protectedwithin a polymeric shell. Coencapsulation of peptides withpeptidase-inhibitors can minimize peptide enzymatic degradation.

The present invention will be further understood by reference to thefollowing non-limiting examples.

EXAMPLE 1 Synthesis of Aerodynamically LightPoly[(p-carboxyphenoxy)-hexane anhydride] ("PCPH") Particles

Aerodynamically light poly[(p-carboxyphenoxy)-hexane anhydride] ("PCPH")particles were synthesized as follows. 100 mg PCPH (MW ˜25,000) wasdissolved in 3.0 mL methylene chloride. To this clear solution was added5.0 mL 1% w/v aqueous polyvinyl alcohol (PVA, MW ˜25,000, 88 mole %hydrolyzed) saturated with methylene chloride, and the mixture wasvortexed (Vortex Genie 2, Fisher Scientific) at maximum speed for oneminute. The resulting milky-white emulsion was poured into a beakercontaining 95 mL 1% PVA and homogenized (Silverson Homogenizers) at 6000RPM for one minute using a 0.75 inch tip. After homogenization, themixture was stirred with a magnetic stirring bar and the methylenechloride quickly extracted from the polymer particles by adding 2 mLisopropyl alcohol. The mixture was continued to stir for 35 minutes toallow complete hardening of the microparticles. The hardened particleswere collected by centrifugation and washed several times with doubledistilled water. The particles were freeze dried to obtain afree-flowing powder void of clumps. Yield, 85-90%.

The mean diameter of this batch was 6.0 μm, however, particles with meandiameters ranging from a few hundred nanometers to several millimetersmay be made with only slight modifications. Scanning electron micrographphotos of a typical batch of PCPH particles showed the particles to behighly porous with irregular surface shape. The particles have a tapdensity less than 0.4 g/cm³.

EXAMPLE 2 Synthesis of PLAL-Lys and PLAL-Lys-Ala Polymeric andCopolymeric Particles

Aerodynamically Light PLAL-Lys Particles

PLAL-Lys particles were prepared by dissolving 50 mg of the graftcopolymer in 0.5 ml dimethylsulfoxide, then adding 1.5 mldichloromethane dropwise. The polymer solution is emulsified in 100 mlof 5% w/v polyvinyl alcohol solution (average molecular weight 25 KDa,88% hydrolyzed) using a homogenizer (Silverson) at a speed ofapproximately 7500 rpm. The resulting dispersion is stirred using amagnetic stirrer for 1 hour. Following this period, the pH is brought to7.0-7.2 by addition of 0.1 N NaOH solution. Stirring is continued for anadditional 2 hours until the methylene chloride is completely evaporatedand the particles hardened. The particles are then isolated bycentrifugation at 4000 rpm (1600 g) for 10 minutes (Sorvall RC-5B). Thesupernatant is discarded and the precipitate washed three times withdistilled water followed by centrifugation for 10 minutes at 4000 rpmeach time. Finally, the particles are resuspended in 5 ml of distilledwater, the dispersion frozen in liquid nitrogen, and lyophilized(Labconco freeze dryer 8) for at least 48 hours. Particle sizing isperformed using a Coulter counter. Average particle mean diametersranged from 100 nm to 14 μm, depending upon processing parameters suchas homogenization speed and time. All particles exhibited tap densitiesless than 0.4 g/cm³. Scanning electron micrograph photos of theparticles showed them to be highly porous with irregular surfaces.

Aerodynamically Light PLAL-Ala-Lys Particles

100 mg of PLAL-Ala-Lys is completely dissolved in 0.4 mltrifluoroethanol, then 1.0 ml methylene chloride is added dropwise. Thepolymer solution is emulsified in 100 ml of 1% w/v polyvinyl alcoholsolution (average molecular weight 25 KDa, 80% hydrolyzed) using asonicator (Sonic & Materal VC-250) for 15 seconds at an output of 40 W.2 ml of 1% PVA solution is added to the mixture and it is vortexed atthe highest speed for 30 seconds. The mixture is quickly poured into abeaker containing 100 ml 0.3% PVA solution, and stirred for three hoursallowing evaporation of the methylene chloride. Scanning electronmicrograph photos of the particles showed them to possess highlyirregular surfaces.

Aerodynamically Light Copolymer Particles

Polymeric aerodynamically light particles consisting of a blend ofPLAL-Lys and PLGA-PEG were made. 50 mg of the PLGA-PEG polymer(molecular weight of PEG: 20 KDa, 1:2 weight ratio of PEG:PLGA, 75:25lactide:glycolide) was completely dissolved in 1 ml dichloromethane. 3mg of poly(lactide-co-lysine)-polylysine graft copolymer is dissolved in0.1 ml dimethylsulfoxide and mixed with the first polymer solution. 0.2ml of TE buffer, pH 7.6, is emulsified in the polymer solution by probesonication (Sonic & Materal VC-250) for 10 seconds at an output of 40 W.To this first emulsion, 2 ml of distilled water is added and mixed usinga vortex mixer at 4000 rpm for 60 seconds. The resulting dispersion isagitated by using a magnetic stirrer for 3 hours until methylenechloride is completely evaporated and microspheres formed. The spheresare then isolated by centrifugation at 5000 rpm for 30 min. Thesupernatant is discarded, the precipitate washed three times withdistilled water and resuspended in 5 ml of water. The dispersion isfrozen in liquid nitrogen and lyophilized for 48 hours.

Variables which may be manipulated to alter the size distribution of theparticles include: polymer concentration, polymer molecular weight,surfactant type (e.g., PVA, PEG, etc.), surfactant concentration, andmixing intensity. Variables which may be manipulated to alter thesurface shape and porosity of the particles include: polymerconcentration, polymer molecular weight, rate of methylene chlorideextraction by isopropyl alcohol (or another miscible solvent), volume ofisopropyl alcohol added, inclusion of an inner water phase, volume ofinner water phase, inclusion of salts or other highly water-solublemolecules in the inner water phase which leak out of the hardeningsphere by osmotic pressure, causing the formation of channels, or pores,in proportion to their concentration, and surfactant type andconcentration.

By scanning electron microscopy (SEM), the PLAL-Lys-PLGA-PEG particleswere highly surface rough and porous. The particles had a mean particlediameter of 7 μm. The blend of PLAL-Lys with poly(lactic acid) (PLA)and/or PLGA-PEG copolymers can be adjusted to adjust particle porosityand size. Additionally, processing parameters such as homogenizationspeed and time can be adjusted. Neither PLAL, PLA nor PLGA-PEG aloneyields an aerodynamically light structure when prepared by thesetechniques.

EXAMPLE 3 Synthesis of Spray-Dried Particles

Aerodynamically Light Particles Containing Polymer and Drug Soluble inCommon Solvent

Aerodynamically light 50:50 PLGA particles were prepared by spray dryingwith testosterone encapsulated within the particles according to thefollowing procedures. 2.0 g poly (D,L-lactic-co-glycolic acid) with amolar ratio of 50:50 (PLGA 50:50, Resomer RG503, B.I. Chemicals,Montvale, N.J.) and 0.50 g testosterone (Sigma Chemical Co., St. Louis,Mo.) are completely dissolved in 100 mL dichloromethane at roomtemperature. The mixture is subsequently spray-dried through a 0.5 mmnozzle at a flow rate of 5 mL/min using a Buchi laboratory spray-drier(model 190, Buchi, Germany). The flow rate of compressed air is 700 nl.The inlet temperature is set to 30° C. and the outlet temperature to 25°C. The aspirator is set to achieve a vacuum of -20 to -25 bar. The yieldis 51% and the mean particle size is approximately 5 μm. Larger particlesize can be achieved by lowering the inlet compressed air flow rate, aswell as by changing other variables. The particles are aerodynamicallylight, as determined by a tap density less than or equal to 0.4 g/cm³.Porosity and surface roughness can be increased by varying the inlet andoutlet temperatures, among other factors.

Aerodynamically Light Particles Containing Polymer and Drug in DifferentSolvents

Aerodynamically light PLA particles with a model hydrophilic drug(dextran) were prepared by spray drying using the following procedure.2.0 mL of an aqueous 10% w/v FITC-dextran (MW 70,000, Sigma ChemicalCo.) solution was emulsified into 100 mL of a 2% w/v solution of poly(D,L-lactic acid) (PLA, Resomer R206, B.I. Chemicals) in dichloromethaneby probe sonication (Vibracell Sonicator, Branson). The emulsion issubsequently spray-dried at a flow rate of 5 mL/min with an air flowrate of 700 nl/h (inlet temperature=30° C., outlet temperature=21° C.,-20 mbar vacuum). The yield is 56%. The particles are aerodynamicallylight, as determined by a tap density less 0.4 g/cm³.

Aerodynamically Light Protein Particles

Aerodynamically light lysozyme particles were prepared by spray dryingusing the following procedure. 4.75 g lysozyme (Sigma) was dissolved in95 mL double distilled water (5% w/v solution) and spray-dried using a0.5 mm nozzle and a Buchi laboratory spray-drier. The flow rate ofcompressed air was 725 nl/h. The flow rate of the lysozyme solution wasset such that, at a set inlet temperature of 97-100° C., the outlettemperature is 55-57° C. The aspirator was set to achieve a vacuum of-30 mbar. The enzymatic activity of lysozyme was found to be unaffectedby this process and the yield of the aerodynamically light particles(tap density less than 0.4 g/cm³) was 66%.

Aerodynamically Light High-Molecular Weight Water-Soluble Particles

Aerodynamically light dextran particles were prepared by spray dryingusing the following procedure. 6.04 g DEAE dextran (Sigma) was dissolvedin 242 mL double distilled water (2.5% w/v solution) and spray-driedusing a 0.5 mm nozzle and a Buchi laboratory spray-drier. The flow rateof compressed air was 750 nl/h. The flow rate of the DEAE-dextransolution was set such that, at a set inlet temperature of 155° C., theoutlet temperature was 80° C. The aspirator was set to achieve a vacuumof -20 mbar. The yield of the aerodynamically light particles (tapdensity less than 0.4 g/cm³) was 66% and the size range ranged between1-15 μm.

Aerodynamically Light Low-Molecular Weight Water-Soluble Particles

Aerodynamically light trehalose particles were prepared by spray dryingusing the following procedure. 4.9 g trehalose (Sigma) was dissolved in192 mL double distilled water (2.5% w/v solution) and spray-dried usinga 0.5 mm nozzle and a Buchi laboratory spray-drier. The flow rate ofcompressed air 650 nl/h. The flow rate of the trehalose solution was setsuch that, at a set inlet temperature of 100° C., the outlet temperaturewas 60° C. The aspirator was set to achieve a vacuum of -30 mbar. Theyield of the aerodynamically light particles (tap density less than 0.4g/cm³) was 36% and the size range ranged between 1-15 μm.

Aerodynamically Light Low-Molecular Weight Water-Soluble Particles

Polyethylene glycol (PEG) is a water-soluble macromolecule, however, itcannot be spray dried from an aqueous solution since it melts at roomtemperatures below that needed to evaporate water. As a result, we havespray-dried PEG at low temperatures from a solution in dichloromethane,a low boiling organic solvent. Aerodynamically light PEG particles wereprepared by spray drying using the following procedure. 5.0 g PEG (MW15,000-20,000, Sigma) was dissolved in 100 mL double distilled water(5.0% w/v solution) and spray-dried using a 0.5 mm nozzle and a Buchilaboratory spray-drier. The flow rate of compressed air 750 nl/h. Theflow rate of the PEG solution was set such that, at a set inlettemperature of 45° C., the outlet temperature was 34-35° C. Theaspirator was set to achieve a vacuum of -22 mbar. The yield of theaerodynamically light particles (tap density less than 0.4 g/cm³) was67% and the size range ranged between 1-15 μm.

EXAMPLE 4 Rhodamine Isothiocyanate Labeling of PLAL and PLAL-LysParticles

Aerodynamically light particles were compared with control particles,referred to herein as "non-light" particles. Lysine amine groups on thesurface of aerodynamically light (PLAL-Lys) and control, non-light(PLAL) particles, with similar mean diameters (6-7 μm) and sizedistributions (standard deviations 34 μm) were labeled with Rhodamineisothiocyanate. The tap density of the porous PLAL-Lys particles was 0.1g/cm³ and that of the non-light PLAL particles was 0.8 g/cm³.

The rhodamine-labeled particles were characterized by confocalmicroscopy. A limited number of lysine functionalities on the surface ofthe solid particle were able to react with rhodamine isothiocyanate, asevidenced by the fluoroscent image. In the aerodynamically lightparticle, the higher lysine content in the graft copolymer and theporous particle structure result in a higher level of rhodamineattachment, with rhodamine attachment dispersed throughout theintersticies of the porous structure. This also demonstrates thattargeting molecules can be attached to the aerodynamically lightparticles for interaction with specific receptor sites within the lungsvia chemical attachment of appropriate targeting agents to the particlesurface.

EXAMPLE 5 Aerosolization of PLAL and PLAL-Lys Particles

To determine whether large aerodynamically light particles can escape(mouth, throat and inhaler) deposition and more efficiently enter theairways and acini than nonporous particles of similar size (referred toherein as non-light or control particles), aerosolization and depositionof aerodynamically light PLAL-Lys (mean diameter 6.3 μm) or control,non-light PLAL (mean diameter 6.9 μm) particles were examined in vitrousing a cascade impactor system.

20 mg of the aerodynamically light or non-light microparticles wereplaced in gelatine capsules (Eli Lilly), the capsules loaded into aSpinhaler dry powder inhaler (DPI) (Fisons), and the DPI activated.Particles were aerosolized into a Mark I Andersen Impactor (AndersenSamplers, Ga.) from the DPI for 30 seconds at 28.3 l/min flow rate. Eachplate of the Andersen Impactor was previously coated with Tween 80 byimmersing the plates in an acetone solution (5% w/vol) and subsequentlyevaporating the acetone in a oven at 60° C. for 5 min. Afteraerosolization and deposition, particles were collected from each stageof the impactor system in separate volumetric flasks by rinsing eachstage with a NaOH solution (0.2 N) in order to completely degrade thepolymers. After incubation at 37° C. for 12 h, the fluorescence of eachsolution was measured (wavelengths of 554 nm excitation, 574 nmemission).

Particles were determined as nonrespirable (mean aerodynamic diameterexceeding 4.7 μm: impactor estimate) if they deposited on the firstthree stages of the impactor, and respirable (mean aerodynamic diameter4.7 μm or less) if they deposited on subsequent stages. FIG. 1 showsthat less than 10% of the non-light (PLAL) particles that exit the DPIare respirable. This is consistent with the large size of themicroparticles and their standard mass density. On the other hand,greater than 55% of the aerodynamically light (PLAL-Lys) particles arerespirable, even though the geometrical dimensions of the two particletypes are almost identical. The lower tap density of the aerodynamicallylight (PLAL-Lys) microparticles is responsible for this improvement inparticle penetration, as discussed further below.

The non-light (PLAL) particles also inefficiently aerosolize from theDPI; typically, less than 40% of the non-light particles exited theSpinhaler DPI for the protocol used. The aerodynamically light(PLAL-Lys) particles exhibited much more efficient aerosolization(approximately 80% of the aerodynamically light microparticles typicallyexited the DPI during aerosolization).

The combined effects of efficient aerosolization and high respirablefraction of aerosolized particle mass means that a far greater fractionof an aerodynamically light particle powder is likely to deposit in thelungs than of a non-light particle powder.

EXAMPLE 6 In Vivo Aerosolization of PLAL and PLAL-Lys Particles

The penetration of aerodynamically light and non-light polymericPLAL-Lys and PLAL microparticles into the lungs was evaluated in an invivo experiment involving the aerosolization of the microparticles intothe airways of live rats.

Male Spraque Dawley rats (150-200 g) were anesthetized using ketamine(90 mg/kg)/xylazine (10 mg/kg). The anesthetized rat was placed ventralside up on a surgical table provided with a temperature controlled padto maintain physiological temperature. The animal was cannulated abovethe carina with an endotracheal tube connected to a Harvard ventilator.The animal was force ventilated for 20 minutes at 300 ml/min. 50 mg ofaerodynamically light (PLAL-Lys) or non-light (PLA) microparticles wereintroduced into the endotracheal tube.

Following the period of forced ventilation, the animal was euthanizedand the lungs and trachea were separately washed using bronchoalveolarlavage. A tracheal cannula was inserted, tied into place, and theairways were washed with 10 ml aliquots of HBSS. The lavage procedurewas repeated until a total volume of 30 ml was collected. The lavagefluid was centrifuged (400 g) and the pellets collected and resuspendedin 2 ml of phenol red-free Hanks balanced salt solution (Gibco, GrandIsland, N.Y.) without Ca² and Mg²⁺ (HBSS). 100 ml were removed forparticle counting using a hemacytometer. The remaining solution wasmixed with 10 ml of 0.4 N NaOH. After incubation at 37° C. for 12 h, thefluorescence of each solution was measured (wavelengths of 554 nmexcitation, 574 nm emission).

FIG. 2 is a bar graph showing total particle mass deposited in thetrachea and after the carina (lungs) in rat lungs and upper airwaysfollowing intratracheal aerosolization during forced ventilation. ThePLAL-Lys aerodynamically light particles had a mean diameter 6.9 μm. Thenon-light PLAL particles had a mean diameter of 6.7 μm. Percent trachealaerodynamically light particle deposition was 54.5, and non-lightdeposition was 77.0. Percent aerodynamically light particle depositionin the lungs was 46.8 and non-light deposition was 23.0.

The non-light (PLAL) particles deposited primarily in the trachea(approximately 79% of all particle mass that entered the trachea). Thisresult is similar to the in vitro performance of the non-lightmicroparticles and is consistent with the relatively large size of thenonlight particles. Approximately 54% of the aerodynamically light(PLAL-Lys) particle mass deposited in the trachea. Therefore, about halfof the aerodynamically light particle mass that enters the tracheatraverses through the trachea and into the airways and acini of the ratlungs, demonstrating the effective penetration of the aerodynamicallylight particles into the lungs.

Following bronchoalveolar lavage, particles remaining in the rat lungswere obtained by careful dissection of the individual lobes of thelungs. The lobes were placed in separate petri dishes containing 5 ml ofHBSS. Each lobe was teased through 60 mesh screen to dissociate thetissue and was then filtered through cotton gauze to remove tissuedebris and connective tissue. The petri dish and gauze were washed withan additional 15 ml of HBSS to maximize microparticle collection. Eachtissue preparation was centrifuged and resuspended in 2 ml of HBSS andthe number of particles counted in a hemacytometer. The particle numbersremaining in the lungs following the bronchoalveolar lavage are shown inFIG. 3. Lobe numbers correspond to: 1) left lung, 2) anterior, 3)median, 4) posterior, 5) postcaval. A considerably greater number ofaerodynamically light PLAL-Lys particles enters every lobe of the lungsthan the nonlight PLAL particles, even though the geometrical dimensionsof the two types of particles are essentially the same. These resultsreflect both the efficiency of aerodynamically light particleaerosolization and the propensity of the aerodynamically light particlesto escape deposition prior to the carina or first bifurcation.

Modifications and variations of the present invention will be obvious tothose skilled in the art from the foregoing detailed description. Suchmodifications and variations are intended to come within the scope ofthe following claims.

What is claimed is:
 1. Biocompatible particles for delivery of atherapeutic, prophylactic or diagnostic agent to the pulmonary systemcomprising a therapeutic, prophylactic or diagnostic agent; wherein theparticles have a tap density less than 0.4 g/cm³ and at least 90% of theparticles have geometric dimensions between about 5 μm and about 30 μm.2. The particles of claim 1 wherein the agent is selected from the groupconsisting of proteins, polysaccharides, lipids, nucleic acids andcombinations thereof.
 3. The particles of claim 1 combined with largebiodegradable carrier particles having a mass mean diameter in the rangeof about 50 μm to about 100 μm.
 4. The particles of claim 1 combinedwith a pharmaceutically acceptable carrier for administration to therespiratory tract.
 5. The particles of claim 1 wherein at least 90% ofthe particles have a mass mean diameter between about 5 μm and about 15μm.
 6. The particles of claim 1 wherein at least 90% of the particleshave a mean diameter between about 9 μm and about 11 μm.
 7. Theparticles of claim 1 wherein at least 50% of the particles have a tapdensity of less than 0.1 g/cm³.
 8. The particles of claim 1 wherein theparticles further comprise a polymeric material.
 9. The particles ofclaim 1 wherein the particles further comprise a non-polymeric material.10. Biocompatible particles for delivery of a targeting molecule to thepulmonary system wherein the targeting molecule is attached to theparticles and wherein the particles have a tap density less than 0.4g/cm³, and at least 90% of the particles have geometric dimensionsbetween 5 μm and about 30 μm.
 11. Biocompatible particles for deliveryof a therapeutic, prophylactic or diagnostic agent to the pulmonarysystem comprising a therapeutic, prophylactic or diagnostic agent;wherein the particles have a tap density less than 0.4 g/cm³ and atleast 90% of the particles have an aerodynamic diameter between about 1μm and about 5 μm.
 12. The particles of claim 11 wherein the agent isselected from the group consisting of proteins, polysaccharides, lipids,nucleic acids and combinations thereof.
 13. The particles of claim 11combined with large biodegradable carrier particles having a mass meandiameter in the range of about 50 μm to about 100 μm.
 14. The particlesof claim 11 combined with a pharmaceutically acceptable carrier foradministration to the respiratory tract.
 15. The particles of claim 11wherein at least 90% of the particles have an aerodynamic diameterbetween about 1 μm and about 3 μm.
 16. The particles of claim 11 whereinat least 90% of the particles have an aerodynamic diameter between about3 μm and about 5 μm.
 17. The particles of claim 11 wherein at least 50%of the particles have a tap density of less than 0.1 g/cm³.
 18. Theparticles of claim 11 wherein the particles further comprise a polymericmaterial.
 19. The particles of claim 11 wherein the particles furthercomprise a non-polymeric material.
 20. Biocompatible particles fordelivery of a targeting molecule to the pulmonary system wherein thetargeting molecule is attached to the particles and wherein theparticles have a tap density less than 0.4 g/cm³, and at least 90% ofthe particles have an aerodynamic diameter between about 1 μm and about5 μm.
 21. A method for delivery of a therapeutic, prophylactic ordiagnostic agent to the pulmonary system comprising:administering to therespiratory tract of a patient in need of treatment, prophylaxis ordiagnosis an effective amount of biocompatible particles, wherein theparticles have a tap density of less than about 0.4 g/cm³ and at least90% of the particles have geometric dimensions between about 5 μm andabout 30 μm.
 22. The method of claim 21 wherein the agent is selectedfrom the group consisting of proteins, polysaccharides, lipids, nucleicacids and combinations thereof.
 23. The method of claim 21 wherein theparticles are combined with large biodegradable carrier particles havinga mass mean diameter in the range of about 50 μm to about 100 μm. 24.The method of claim 21 wherein the particles are combined with apharmaceutically acceptable carrier for administration to therespiratory tract.
 25. The method of claim 21 wherein at least 90% ofthe particles have a mass mean diameter between about 5 μm and about 15μm.
 26. The method of claim 21 for delivery to the alveolar zone of thelung wherein at least 90% of the particles have a mean diameter betweenabout 9 and about 11 μm.
 27. The method of claim 21 wherein at least 50%of the administered particles have a tap density of less than about 0.1g/cm³.
 28. The method of claim 21 wherein the particles further comprisea polymeric material.
 29. The method of claim 21 wherein the particlesfurther comprise a non-polymeric material.
 30. A method for delivery ofa targeting molecule to the pulmonary system comprising:administering tothe respiratory tract of a patient in need of treatment, prophylaxis ordiagnosis an effective amount of biocompatible particles, wherein theparticles have a tap density less than about 0.4 g/cm³ and at least 90%of the particles have geometric dimensions between about 5 μm and about30 μm, and wherein the targeting molecule is attached to the particles.31. A method for delivery of a therapeutic, prophylactic or diagnosticagent to the pulmonary system comprising:administering to therespiratory tract of a patient in need of treatment, prophylaxis ordiagnosis an effective amount of biocompatible particles, wherein theparticles have a tap density of less than about 0.4 g/cm³ and at least90% of the particles have an aerodynamic diameter between about 1 μm andabout 5 μm.
 32. The method of claim 31 wherein the agent is selectedfrom the group consisting of proteins, polysaccharides, lipids, nucleicacids and combinations thereof.
 33. The method of claim 31 wherein theparticles are combined with large biodegradable carrier particles havinga mass mean diameter in the range of about 50 μm to about 100 μm. 34.The method of claim 31 wherein the particles are combined with apharmaceutically acceptable carrier for administration to therespiratory tract.
 35. The method of claim 31 wherein at least 90% ofthe particles have an aerodynamic diameter between about 1 μm and about3 μm.
 36. The method of claim 31 for delivery to the alveolar zone ofthe lung wherein at least 90% of the particles have an aerodynamicdiameter between about 3 μm and about 5 μm.
 37. The method of claim 31wherein at least 50% of the administered particles have a tap density ofless than about 0.1 g/cm³.
 38. The method of claim 31 wherein theparticles further comprise a polymeric material.
 39. The method of claim31 wherein the particles further comprise a non-polymeric material. 40.A method for delivery of a targeting molecule to the pulmonary systemcomprising:administering to the respiratory tract of a patient in needof treatment, prophylaxis or diagnosis an effective amount ofbiocompatible particles, wherein the particles have a tap density lessthan about 0.4 g/cm³ and at least 90% of the particles have anaerodynamic diameter between about 1 μm and about 5 μm, and wherein thetargeting molecule is attached to the particles.